MicroRNA deregulation is a consistent feature of glioblastoma, yet the biological effect of each single gene is generally modest, and therapeutically negligible. Here we describe a module of ...microRNAs, constituted by miR-124, miR-128 and miR-137, which are co-expressed during neuronal differentiation and simultaneously lost in gliomagenesis. Each one of these miRs targets several transcriptional regulators, including the oncogenic chromatin repressors EZH2, BMI1 and LSD1, which are functionally interdependent and involved in glioblastoma recurrence after therapeutic chemoradiation. Synchronizing the expression of these three microRNAs in a gene therapy approach displays significant anticancer synergism, abrogates this epigenetic-mediated, multi-protein tumor survival mechanism and results in a 5-fold increase in survival when combined with chemotherapy in murine glioblastoma models. These transgenic microRNA clusters display intercellular propagation in vivo, via extracellular vesicles, extending their biological effect throughout the whole tumor. Our results support the rationale and feasibility of combinatorial microRNA strategies for anticancer therapies.
We sought a novel approach against glioblastomas (GBM) focused on targeting signaling molecules localized in the tumor extracellular matrix (ECM). We investigated fibulin-3, a glycoprotein that forms ...the ECM scaffold of GBMs and promotes tumor progression by driving Notch and NFκB signaling.
We used deletion constructs to identify a key signaling motif of fibulin-3. An mAb (mAb428.2) was generated against this epitope and extensively validated for specific detection of human fibulin-3. mAb428.2 was tested in cultures to measure its inhibitory effect on fibulin-3 signaling. Nude mice carrying subcutaneous and intracranial GBM xenografts were treated with the maximum achievable dose of mAb428.2 to measure target engagement and antitumor efficacy.
We identified a critical 23-amino acid sequence of fibulin-3 that activates its signaling mechanisms. mAb428.2 binds to that epitope with nanomolar affinity and blocks the ability of fibulin-3 to activate ADAM17, Notch, and NFκB signaling in GBM cells. mAb428.2 treatment of subcutaneous GBM xenografts inhibited fibulin-3, increased tumor cell apoptosis, and enhanced the infiltration of inflammatory macrophages. The antibody reduced tumor growth and extended survival of mice carrying GBMs as well as other fibulin-3-expressing tumors. Locally infused mAb428.2 showed efficacy against intracranial GBMs, increasing tumor apoptosis and reducing tumor invasion and vascularization, which are enhanced by fibulin-3.
To our knowledge, this is the first rationally developed, function-blocking antibody against an ECM target in GBM. Our results offer a proof of principle for using "anti-ECM" strategies toward more efficient targeted therapies for malignant glioma.
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BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic ...strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR-Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers.
The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated miRNAs rather than the action of a single miRNA. ...The characterization of these specific miRNAs modules is a fundamental step in maximizing their use in therapy. This is extremely relevant because their combinatorial attributes can be practically exploited. Described here is a method to define a specific miRNA signature relevant to the control of oncogenic chromatin repressors in glioblastoma. The approach first defines a general group of miRNAs that are deregulated in tumors in comparison to normal tissue. The analysis is further refined by differential culture conditions, underscoring a subgroup of miRNAs that are co-expressed simultaneously during specific cellular states. Finally, the miRNAs that satisfy these filters are combined into an artificial polycistronic transgenes, which is based on a scaffold of naturally existing miRNA clusters genes, then used for overexpression of these miRNA modules into receiving cells.
The cellular machinery regulating microRNA biogenesis and maturation relies on a small number of simple steps and minimal biological requirements and is broadly conserved in all eukaryotic cells. The ...same holds true in disease. This allows for a substantial degree of freedom in the engineering of transgenes capable of simultaneously expressing multiple microRNAs of choice, allowing a more comprehensive modulation of microRNA landscapes, the study of their functional interaction, and the possibility of using such synergism for gene therapy applications. We have previously engineered a transgenic cluster of functionally associated microRNAs to express a module of suppressed microRNAs in brain cancer for therapeutic purposes. Here, we provide a detailed protocol for the design, cloning, delivery, and utilization of such artificial microRNA clusters for gene therapy purposes. In comparison with other protocols, our strategy effectively decreases the requirements for molecular cloning, because the nucleic acid sequence encoding the combination of the desired microRNAs is designed and validated in silico and then directly synthesized as DNA that is ready for subcloning into appropriate delivery vectors, for both in vitro and in vivo use. Sequence design and engineering require 4-5 h. Synthesis of the resulting DNA sequence requires 4-6 h. This protocol is quick and flexible and does not require special laboratory equipment or techniques, or multiple cloning steps. It can be easily executed by any graduate student or technician with basic molecular biology knowledge.
Glioblastoma (GBM) is resistant to standard of care. Immune checkpoints inhibitors (such as anti-PD-1 mAbs) efficiently restore antitumor T-cell activity. We engineered a new oncolytic herpes simplex ...virus (oHSV) expressing a single-chain antibody against PD-1 (scFvPD-1) to evaluate its efficacy in mouse models of GBM.
NG34scFvPD-1 expresses the human
gene transcriptionally controlled by the Nestin promoter to allow replication in GBM cells and a scFvPD-1 cDNA transcriptionally controlled by the CMV promoter. ELISA assays were performed to detect binding of scFvPD-1 to mouse and human PD-1.
cytotoxicity and replication assays were performed to measure NG34scFvPD-1 oncolysis, and scFvPD-1 expression and secretion were determined.
survival studies using orthotopic mouse GBM models were performed to evaluate the therapeutic potency of NG34scFvPD-1.
NG34scFvPD-1-infected GBM cells express and secrete scFvPD-1 that binds mouse PD-1. The introduction of the scFvPD-1 sequence in the viral backbone does not alter the oncolytic properties of NG34scFvPD-1.
NG34scFvPD-1 treatment improved the survival with a tail of durable survivorship in 2 syngeneic immunocompetent mouse models of GBM. Mice that survived the first GBM challenge rejected the second challenge of GBM when implanted in the contralateral hemisphere. However, this was not true when athymic mice were employed as the recipients of the second challenge, consistent with the need for an intact immune system to obtain a memory response.
NG34scFvPD-1 treatment induces a durable antitumor response in 2 preclinical mouse models of GBM with evidence for antitumor memory.
Molecular profiling of glioblastomas has revealed the presence of key signaling hubs that contribute to tumor progression and acquisition of resistance. One of these main signaling mechanisms is the ...nuclear factor-kappa B (NF-κB) pathway, which integrates multiple extracellular signals into transcriptional programs for tumor growth, invasion and maintenance of the tumor-initiating population. We show here that an extracellular protein released by glioblastoma cells, fibulin-3, drives oncogenic NF-κB in the tumor and increases NF-κB activation in peritumoral astrocytes. Fibulin-3 expression correlates with a NF-κB-regulated 'invasive signature' linked to poorer survival, being a possible tissue marker for regions of active tumor progression. Accordingly, fibulin-3 promotes glioblastoma invasion in a manner that requires NF-κB activation both in the tumor cells and their microenvironment. Mechanistically, we found that fibulin-3 activates the metalloprotease ADAM17 by competing with its endogenous inhibitor, TIMP3. This results in sustained release of soluble tumor necrosis factor alpha (TNFα) by ADAM17, which in turn activates TNF receptors and canonical NF-κB signaling. Taken together, our results underscore fibulin-3 as a novel extracellular signal with strong activating effect on NF-κB in malignant gliomas. Because fibulin-3 is produced de novo in these tumors and is absent from the normal brain, we propose that targeting the fibulin-3/NF-κB axis may provide a novel avenue to disrupt oncogenic NF-κB signaling in combination therapies for malignant brain tumors.
The cell-surface metalloprotease ADAM17/TACE regulates multiple signaling cascades, such as Notch and NFkB, which are critical for glioblastoma (GBM) growth and invasion. This enzyme is tonically ...inhibited by the extracellular matrix (ECM)-associated protein TIMP3, but signals that release ADAM17 inhibition have not been described. Fibulin-3 is an ECM protein highly enriched in GBMs that activates Notch and NFkB signaling but does not seem to bind to membrane receptors for these pathways. Therefore, we investigated if fibulin-3 could simultaneously regulate both mechanisms in GBM by activating ADAM17. Using cultured GBM cells we demonstrated that fibulin-3 increases the expression and activity of ADAM17. Activated ADAM17 released membrane-bound TNF-alpha, which activated TNF-alpha receptors and NFkB signaling. At the same time activated ADAM17 increased the cleavage and activity of Notch1 receptor, explaining the ability of fibulin-3 to simultaneously regulate multiple pro-tumoral pathways. Using mixed cultures, ex vivo brain slices, and intracranial tumor models, we demonstrated that fibulin-3 secreted by GBM cells also increases Notch activity in endothelial cells to enhance angiogenesis, and NFkB activity in astroglial cells to facilitate tumor invasion. Mechanistically, fibulin-3 regulated ADAM17 by competition with TIMP3; accordingly, downregulation of fibulin-3 reduced ADAM17 activity and silenced Notch and NFkB signaling in GBM cells. We identified an N-terminal sequence of fibulin-3 that was critical to capture TIMP3 and activate ADAM17. An antibody against this sequence blocked fibulin-3 and downregulated ADAM17 activity in GBMs, reducing tumor growth. In conclusion, ADAM17 is critical to drive pro-tumoral programs in GBMs, and is in turn regulated by competition of stimulatory (fibulin-3) and inhibitory (TIMP3) signals in the tumor ECM. Disrupting the fibulin-3/ADAM17 axis could inhibit multiple pro-tumoral mechanisms and cause widespread anti-tumor effects.
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